Characterization of a New Cyclohexylamine Oxidase From Acinetobacter

Cyclohexylamine (CHAM) is widely used in various industries, but it is harmful to human beings and the environment. Acinetobacter sp. YT-02 can degrade CHAM via cyclohexanone as an intermediate. In this study, the cyclohexylamine oxidase (CHAO) gene from Acinetobacter sp. YT-02 was cloned. Amino acid sequence alignment indicated that the cyclohexylamine oxidase (CHAOYT–02) was 48% identical to its homolog from Brevibacterium oxydans IH-35A (CHAOIH–35). The enzyme was expressed in Escherichia coli BL21 (DE3), and purified to apparent homogeneity by Ni-affinity chromatography. The purified enzyme was proposed to be a dimer of molecular mass of approximately 91 kDa. The enzyme exhibited its maximum activity at 50°C and at pH 7.0. The enzyme was thermolabile as demonstrated by loss of important percentage of its maximal activity after 30 min incubation at 50°C. Metal ions Mg2+, Co2+, and K+ had certain inhibitory effect on the enzyme activity. The kinetic parameters Km and Vmax were 0.25 ± 0.02 mM and 4.3 ± 0.083 μM min?1, respectively. The biochemical properties, substrate specificities, and three-dimensional structures of CHAOYT–02 and CHAOIH–35 were compared. Our results are helpful to elucidate the mechanism of microbial degradation of CHAM in the strain YT-02. In addition, CHAOYT–02, as a potential biocatalyst, is promising in controlling CHAM pollution and deracemization of chiral amines.

Cyclohexylamine (CHAM), an important fine chemical intermediate, is widely used in industrial manufaction of insecticides, antiseptics and plasticizers. In the process of production and usage, CHAM is released into the atmosphere, water, and soil. Furthermore, It was found that sodium N-cyclohexylsulfamate, which is used as a food additive and produced 100,000 tons annually in China, could be decomposed into CHAM by intestinal bacteria. CHAM is classified as a volatile organic compound that can enter human body via inhalation or skin contact. CHAM has drawn increasing attention as a weak carcinogen. To eliminate CHAM residual dispersed in the environment, certain measures have to be taken. Microbial degradation has advantages of low cost and less energy consumption and is an effective approach to eliminating CHAM pollution and residues in the environment.

To date, limited studies showed that bacteria utilized CHAM as the only source of carbon and nitrogen. These bacteria included Brevibacterium oxydans IH-35A. Pseudomonas plecoglossicida NyZ12, and Acinetobacter sp. YT-02 isolated by our group. Acinetobacter sp. YT-02 is a Gram-negative bacterium isolated from the activated sludge from a sodium N-cyclohexylsulfamate production plant. The draft genome showed that Acinetobacter sp. YT-02 had poor similarity with cyclohexylamine-degrading bacteria P. plecoglossicida NyZ12, indicating that our isolate is a new member of CHAM-degrading bacteria. These strains can convert CHAM into cyclohexanone which was catalyzed by cyclohexylamine oxidase, a type of monoamine oxidases (MAOs). CHAO catalyzes the oxidative deaminnation of cyclohexylamine to form cyclohexanone, hydrogen peroxide and ammonia, using FAD as cofactor. To date, only CHAO gene from strain B. oxydans IH-35A has been cloned, and an X-ray structure of CHAO from B. oxydans IH-35A (CHAOIH–35A) was determined. Although CHAO derived from Pseudomonas plecoglossicida NyZ12 was confirmed to be involved in the degradation of cyclohexylamine, there were no relevant biochemical properties reported for this enzyme. The related biochemical properties for CHAO from Pseudomonas sp. (purified enzyme) were investigated, but there is no relevant genetic information. The aim of this study is to clone and heterologously express the CHAO gene from Acinetobacter sp. YT-02. Moreover, the enzymatic properties, kinetic parameters, and three-dimensional structure of Acinetobacter sp. YT-02 (CHAOYT–02) were investigated. The results indicated that CHAOYT–02 could be used for elimination of cyclohexylamine in the environment. In addition, CHAOYT–02, as a potential biocatalyst in the deracemization of chiral amines, is promising in pharmaceutical industry.